US4302480A - Thin cover sheet for use in microscopic staining and a process for its production - Google Patents

Thin cover sheet for use in microscopic staining and a process for its production Download PDF

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Publication number
US4302480A
US4302480A US06/049,010 US4901079A US4302480A US 4302480 A US4302480 A US 4302480A US 4901079 A US4901079 A US 4901079A US 4302480 A US4302480 A US 4302480A
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US
United States
Prior art keywords
cover sheet
colorant
polymer
thin cover
staining
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US06/049,010
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English (en)
Inventor
Wolfgang Fischer
Brigitte Wissel
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Merck Patent GmbH
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Merck Patent GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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Assigned to MERCK PATENT GESELLSCHAFT MIT BESCHRANKTER HAFTUNG reassignment MERCK PATENT GESELLSCHAFT MIT BESCHRANKTER HAFTUNG ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: FISCHER WOLFGANG, WISSEL, BRIGITTE
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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/31504—Composite [nonstructural laminate]

Definitions

  • the present invention relates to a new thin cover sheet for use in microscopic staining methods and to a new process for its production.
  • a thin cover sheet for use in microscopic staining consisting essentially of a transparent, water-insoluble plastic carrier and a coating thereon comprising a colorant, and at least one polymer which is soluble both in water and in lower aliphatic alcohols.
  • these objects have been attained by providing a process for producing the above thin cover sheet comprising applying at least one layer of an aqueous-alcoholic solution of the colorant and of the polymer to the plastic carrier, and drying the resulting coating.
  • the present invention also relates to the use of the thin cover sheet for staining when carrying out microscopic investigations.
  • the incorporation of specific organic polymers improves the quality of the dye solutions and dye coatings to such an extent that the difficulties mentioned can be regarded as having been overcome.
  • the dye solutions of this invention are also suitable for application by printing techniques.
  • Stainings which have been carried out using the thin cover sheets according to this invention offer the same differentiation possibilities as the standard methods conventional hitherto, which were carried out using dyestuff solutions, and are virtually indistinguishable from these.
  • the advantages of the new stainings include the fact that no separate equipment, for example a staining stage or automatic staining device is required for staining. Further, the irksome handling of dye solutions is dispensed with and staining can always be carried out with the same dyestuff composition. Only per this invention, however, can the standardization desirable in a practical laboratory be achieved.
  • the process according to this invention is carried out by adding a suitable solvent (for example water or a lower alcohol) to the desired dyestuffs; adding buffer substances or tensides if desired; adding a specific amount of an organic polymer; and dissolving all the constituents of the mixture with stirring.
  • a suitable solvent for example water or a lower alcohol
  • the resulting solution is applied to the thin sheet and dried, for example by air and/or by supplying heat, to give a thin film on the thin sheet.
  • a sheet-like carrier made of fibrous material which contains the color reagent in a state of fine division, is described in U.S. Pat. Spec. No. 3,678,151.
  • the carrier is dipped into a solution and placed on the microscope slide, to which the sample has been applied. After staining, the carrier is removed and the sample is examined under the microscope.
  • the thin cover sheet according to this invention does not consist of fibrous material; also, it contains the colorant only in the layer applied to the surface and is therefore of a different composition.
  • rigid thin transfer sheets for chemical analysis on a microscopically small scale are described in Chemiker-Zeitung 97, 200 to 205 (1973). These thin sheets consist of gelatine or polyvinyl alcohol and contain the detection reagent incorporated therein. In a particular embodiment, these thin sheets are applied to a rigid carrier, for example a carrier made of polyester, and form a thin laminate together with this carrier. In contrast to this, the coating of the thin cover sheet according to this invention contains a polymer which is soluble both in water and in lower aliphatic alcohols. This does not form a thin sheet and is not suitable for reproduction analysis.
  • a thin cover sheet is understood to mean a thin sheet which is made of a plastic which is more or less elastic and which can be placed on a microscope slide.
  • the colorant coating of the thin cover sheet according to this invention is so arranged that it comes into contact with the biological sample to be stained, on the microscope slide.
  • the shape of the thin cover sheet is capable of further variation and can be, for example, round or polygonal. Usually, the shape corresponds approximately to that of the microscope slide customarily used, but it can also be smaller.
  • the thickness of the thin cover sheet can be 0.01 to 0.5 mm but is preferably about 0.05 to 0.2 mm.
  • the material from which the thin cover sheet is made is an appropriately transparent, water-insoluble plastic. It must be of a nature such that as far as possible no reagents are absorbed therein or held fast thereto. Therefore, for example, absorbent carriers, such as are used for reagent papers, cannot be used. In other respects, however, the choice of the material for the thin sheet is not critical.
  • suitable transparent, water-insoluble plastics are polymers and polycondensation products, such as polyvinyl chloride (PVC), polyesters, polyamides, polycarbonates, polystyrene, polyurethanes, polyalkenes, such as polyethylene or polypropylene, polyethylene terephthalate or other high molecular weight organic substances, such as cellulose acetate.
  • PVC polyvinyl chloride
  • polyesters such as polyethylene or polypropylene, polyethylene terephthalate or other high molecular weight organic substances, such as cellulose acetate.
  • PVC polyvinyl chloride
  • polyesters such as polyamides, polycarbonates, polystyrene, polyurethanes, polyalkenes, such as polyethylene or polypropylene, polyethylene terephthalate or other high molecular weight organic substances, such as cellulose acetate.
  • the coating on the thin cover sheet according to the invention has the thinness of a film and can partially or--preferably--entirely cover the side of the thin cover sheet which faces towards the microscope slide.
  • the thickness of the coating can be 0.01 to 0.02 mm and is preferably 0.05-0.15 mm.
  • Polymers suitable for the coating include all polymers and copolymers of various degrees of polymerization which appear transparent on moistening and are soluble both in water and in lower aliphatic alcohols; for example preferably solid and semi-solid homopolymers of vinylpyrrolidone and vinyl alcohol units; and also polyglycols of units of, for example, ethylene glycol or corresponding copolymers of these and other monomers in various molar ratios, for example copolymers of vinylpyrrolidone and vinyl acetate.
  • Polylactides and corresponding copolymers with the said monomers are also suitable, for example copolymers of lactide and glycolide in various molar ratios.
  • lower aliphatic alcohols include, preferably, aliphatic alcohols with up to 6 C atoms, for example methanol, ethanol, propanol, isopropanol, butanol, isobutanol, sec-butanol, tert-butanol, pentanol, hexanol or isohexanol.
  • the solution of the colorant, with the aid of which the coating is produced, and the coating itself can also contain further additives, for example emulsifiers.
  • Suitable additives are, in particular, ionic or especially non-ionic tensides.
  • These substances can consist, for example, of oxalkylated higher fatty alcohols, for example oxethylated or oxpropylated fatty alcohols with a chain length of 10-20 C atoms; alcohols of this type which can be used are preferably the natural or synthetically accessible, saturated or unsaturated straight-chain alcohols, for example various fractions of coconut fatty alcohol or the fatty alcohols accessible by special hydrogenation of unsaturated fatty acids.
  • Tensides which can be used are appropriately also oxalkylated higher alkylphenols, for example oxethylated octyl-, nonyl- or decyl- phenols, and the oxethylated nonylphenols which are available commercially are preferred.
  • Colorants which can be employed are, in principle, all dyestuffs and all reagents which give a characteristic coloration with the samples to be detected.
  • suitable colorants are those substances which with specific biological samples, such as, for example, constituents of the blood, of the tissue, of the connective tissue, various glands and organs, the bone marrow, the bone, the body fat, glycogen and collagen, antibodies, mucuses (for example mucuses of the uterus or of the vagina) or sugars or constituents of plants, micro-organisms (for example bacteria) or protozoa give one of the color-forming agents known from the literature, such as are described in detail, for example, in "Staining Procedures", 3rd edition, edited by George Clark, The Williams & Wilkens Company, Baltimore/U.S.A.
  • the thin cover sheets of this invention are especially suitable for blood staining according to Giemsa, May-Grunwald or Wright, for supravital staining, for bacteria staining according to Gram, for the Ziehl-Neelsen stain for staining acid-resistant bacteria or for the Papanicolaou stain for the examination of specific mucuses (for example vaginal mucus).
  • the ratio of the amounts of colorant and polymer in the dye solution and--derivable therefrom--in the coating on the thin sheet can vary within wide limits and is in particular dependent on the viscosity of the solution of the colorant and of the polymer to be employed.
  • more polymer substance is always necessary if the viscosity of the solution of the second polymer at the same concentration is lower than that of the solution of the first polymer.
  • the amount of organic polymer in the coating of the thin sheet is 0.05-5 g/m 2 of the sheet.
  • concentration of the organic polymer is preferably about 0.1-10%; however, these values can vary greatly, depending on the colorant used.
  • the coating preferably contains about 1-30% by weight of colorant and especially about 10-20% by weight; however, variations outside these values are again possible, depending on the colorant used.
  • the solutions of the colorant can be applied to the thin sheet by various methods. Thus, it is possible uniformly to distribute the solution manually, for example using a sponge, on the thin sheet. In place of a sponge, an artist's brush, a cloth or a soft flat brush are also advantageous. Application of the colorant by spraying is also possible. In practice, however, application of the colorant by processes used in printing technology is particularly preferred, for example by the processes, which are in themselves known, of photogravure printing, screen printing or application with rollers. In the case of printing processes with rollers, the dye solution is in the holes of a printing roller which, as a result of suitable measures (wiping off), is otherwise free from dyestuff, and can be transferred to the material of the thin sheet by rotating the roller.
  • At least one application of the dye solution is made to the thin cover sheet; 5 applications are, for example, certainly possible. However, 3 applications are the rule and are usually adequate for the intended purposes.
  • the viscosity of the final dye solution is about 1-1,000 and preferably 25-500 centipoise.
  • the solution of the dye without the polymer is thickened to such an extent by the addition of the polymer that even solutions in which the colorant is in low concentration can be applied as a uniform coating to the thin sheets.
  • the coating is dried in a conventional manner, for example, in a stream of air and/or by the action of heat or infrared light. Appropriately, drying is carried out in a stream of air at 50°-60° C.
  • the thin cover sheet of this invention When a stain produced using the thin cover sheet of this invention is evaluated under a microscope, the thin cover sheet can be present or absent, as desired. If staining merely for general information purposes or staining for routine purposes is desired, the thin sheet can be left on the microscope slide when evaluating, for the sake of simplicity. If, on the other hand, it is desired to store the stain, for example for documentation purposes, then, for example, the thin sheet can be removed before evaluating and the stained sample will be washed and then dried.
  • Dye solution is transferred to a thin sheet of polycarbonate from a rotating roller which is continuously dipped into an ethanolic dyestuff solution of Giemsa's azure-eosin-methylene blue prepared analogously to Example 1(a) and any excess of dye solution which may arise is removed using a doctor blade. The thin sheet is then dried and cut into suitable pieces.
  • a drop of blood flowing from the ear lobe or finger tip is applied to a cleaned microscope slide and dispersed in the customary manner using a cover slip held at an angle, by drawing the drop of blood along with the cover slip.
  • the blood smear is dried in air and fixed by dipping in methanol or ethanol of 10 seconds. It is then dried.
  • One drop of a buffer solution of pH 6 is now applied to the center of the blood smear and the thin cover sheet according to Example 1(a), (b) or (c) is placed with its dye coating on the blood smear for 10 minutes at room temperature. During this time the buffer solution spreads uniformly in a thin layer between the thin sheet and the smear.
  • the thin dye sheet is then removed and the microscope slide is rinsed with distilled water at pH 7.2 (or with a Weise buffer solution) and dried in air.
  • the blood picture is evaluated under a microscope.
  • the stained blood smear produced in this way can be stored for a relatively long time.
  • a thin sheet of polyvinyl chloride is coated with a dyestuff solution which has the following composition: 0.14 kg of May-Grunwald's eosin-methylene-blue, 1.25 kg of vinylpyrrolidone/vinyl acetate copolymer (Luviskol® K 90), 0.30 liter of a 1% aqueous solution of a nonylphenol polyglycol ether (Marlophen®) and 50 liters of ethanol.
  • a dyestuff solution which has the following composition: 0.14 kg of May-Grunwald's eosin-methylene-blue, 1.25 kg of vinylpyrrolidone/vinyl acetate copolymer (Luviskol® K 90), 0.30 liter of a 1% aqueous solution of a nonylphenol polyglycol ether (Marlophen®) and 50 liters of ethanol.
  • a thin sheet of polyvinyl chloride is coated with a dyestuff solution which has the following composition: 0.12 kg of Wright's eosin-methylene-blue, 1.25 kg of a vinylpyrrolidone/vinyl acetate copolymer (Luviskol® K 90), 0.32 liter of a 1% aqueous solution of a fatty alcohol polyglycol ether (Marlipal®) and 50 liters of ethanol.
  • a dyestuff solution which has the following composition: 0.12 kg of Wright's eosin-methylene-blue, 1.25 kg of a vinylpyrrolidone/vinyl acetate copolymer (Luviskol® K 90), 0.32 liter of a 1% aqueous solution of a fatty alcohol polyglycol ether (Marlipal®) and 50 liters of ethanol.

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  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Optics & Photonics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Paints Or Removers (AREA)
  • Microscoopes, Condenser (AREA)
US06/049,010 1978-06-16 1979-06-15 Thin cover sheet for use in microscopic staining and a process for its production Expired - Lifetime US4302480A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2826363A DE2826363C2 (de) 1978-06-16 1978-06-16 Deckfolie zur Verwendung in mikroskopischen Färbeverfahren und Verfahren zu ihrer Herstellung
DE2826363 1978-06-16

Publications (1)

Publication Number Publication Date
US4302480A true US4302480A (en) 1981-11-24

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US06/049,010 Expired - Lifetime US4302480A (en) 1978-06-16 1979-06-15 Thin cover sheet for use in microscopic staining and a process for its production

Country Status (11)

Country Link
US (1) US4302480A (de)
JP (1) JPS552988A (de)
BE (1) BE876970A (de)
BR (1) BR7903793A (de)
CH (1) CH641726A5 (de)
DE (1) DE2826363C2 (de)
FR (1) FR2428853A1 (de)
GB (1) GB2023871B (de)
IT (1) IT1117185B (de)
NL (1) NL7904721A (de)
SE (1) SE7905297L (de)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4493821A (en) * 1982-02-04 1985-01-15 Harrison James S Preservative and fixative preparations for biological systems
US4578282A (en) * 1982-02-04 1986-03-25 Harrison James S Composition for diagnostic reagents
US4597982A (en) * 1982-03-18 1986-07-01 Delameter William D Method for processing flexible sheets
US4853262A (en) * 1985-08-13 1989-08-01 Fuji Photo Film Co., Ltd. Cover film for microscopy
US5219557A (en) * 1986-12-24 1993-06-15 Merck Patent Gesellschaft Mit Beschrankter Haftung Covering agent for microscopy specimens
WO2001033279A1 (fr) * 1999-10-27 2001-05-10 Muto Pure Chemicals Co., Ltd Lamelle couvre-objet sur laquelle est imprime un support pour preparations microscopiques et son procede de production
US6239906B1 (en) 1997-09-04 2001-05-29 Andrew E. Lorincz Flexible microscope slide
US6268208B1 (en) * 1998-02-18 2001-07-31 Sysmex Corporation Drying configuration and method in smear specimen preparation system
US6495195B2 (en) 1997-02-14 2002-12-17 Arcturus Engineering, Inc. Broadband absorbing film for laser capture microdissection
US6567214B2 (en) * 1997-09-04 2003-05-20 Andrew E. Lorincz Microscope slide having culture media and method for use
US20040058101A1 (en) * 2000-09-28 2004-03-25 Jorg Klemm Method and device for producing singulet oxygen
US20040121339A1 (en) * 2002-12-19 2004-06-24 Jizhong Zhou Special film-coated substrate for bio-microarray fabrication and use thereof
WO2005085799A1 (de) * 2004-03-04 2005-09-15 Merck Patent Gmbh Mittel zum eindecken von mikroskopischen präparaten
US7075640B2 (en) 1997-10-01 2006-07-11 Arcturus Bioscience, Inc. Consumable for laser capture microdissection
WO2007084429A2 (en) * 2006-01-13 2007-07-26 Ventana Medical Systems, Inc. Biological sample processing composition and method
US20080261266A1 (en) * 2004-12-17 2008-10-23 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
CN111175102A (zh) * 2020-01-13 2020-05-19 沈阳农业大学 一种芍药属植物根尖染色体制片的方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61254916A (ja) * 1985-05-07 1986-11-12 Mitsubishi Rayon Co Ltd 顕微鏡用カバ−グラス
GB2333358A (en) * 1998-01-15 1999-07-21 Binding Site Ltd Reagent delivery to a sample site

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US3678151A (en) * 1969-07-25 1972-07-18 Gugol Clini Tex Inc Biological staining method
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US3796594A (en) * 1970-10-30 1974-03-12 Gen Electric Stain coated slides for differentially staining blood
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US3768978A (en) * 1971-03-01 1973-10-30 Hamilton Co Disposable pipette
US3980436A (en) * 1974-12-19 1976-09-14 Sci-Med Laboratories, Inc. Analytic test device
US3937613A (en) * 1975-04-04 1976-02-10 Alpha Chemicals (Australia) Pty. Limited Device for the determination of chlorine in the water of swimming pools
US4103041A (en) * 1975-04-11 1978-07-25 Boehringer Mannheim Gmbh Process for coating slides

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Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4578282A (en) * 1982-02-04 1986-03-25 Harrison James S Composition for diagnostic reagents
US4493821A (en) * 1982-02-04 1985-01-15 Harrison James S Preservative and fixative preparations for biological systems
US4597982A (en) * 1982-03-18 1986-07-01 Delameter William D Method for processing flexible sheets
US4853262A (en) * 1985-08-13 1989-08-01 Fuji Photo Film Co., Ltd. Cover film for microscopy
US5219557A (en) * 1986-12-24 1993-06-15 Merck Patent Gesellschaft Mit Beschrankter Haftung Covering agent for microscopy specimens
US6495195B2 (en) 1997-02-14 2002-12-17 Arcturus Engineering, Inc. Broadband absorbing film for laser capture microdissection
US6239906B1 (en) 1997-09-04 2001-05-29 Andrew E. Lorincz Flexible microscope slide
US6567214B2 (en) * 1997-09-04 2003-05-20 Andrew E. Lorincz Microscope slide having culture media and method for use
US7221447B2 (en) 1997-10-01 2007-05-22 Molecular Devices Corporation Consumable for laser capture microdissection
US7075640B2 (en) 1997-10-01 2006-07-11 Arcturus Bioscience, Inc. Consumable for laser capture microdissection
US6268208B1 (en) * 1998-02-18 2001-07-31 Sysmex Corporation Drying configuration and method in smear specimen preparation system
WO2001033279A1 (fr) * 1999-10-27 2001-05-10 Muto Pure Chemicals Co., Ltd Lamelle couvre-objet sur laquelle est imprime un support pour preparations microscopiques et son procede de production
US6991831B2 (en) * 2000-09-28 2006-01-31 Natural Energy Solutions, Ag Method and device for producing singlet oxygen
US20040058101A1 (en) * 2000-09-28 2004-03-25 Jorg Klemm Method and device for producing singulet oxygen
US20050048554A1 (en) * 2002-12-19 2005-03-03 Jizhong Zhou Method of making and using hybrid polymeric thin films for bio-microarray applications
US20040121339A1 (en) * 2002-12-19 2004-06-24 Jizhong Zhou Special film-coated substrate for bio-microarray fabrication and use thereof
WO2005085799A1 (de) * 2004-03-04 2005-09-15 Merck Patent Gmbh Mittel zum eindecken von mikroskopischen präparaten
US20070166197A1 (en) * 2004-03-04 2007-07-19 Guenter Hauke Means for covering microscopic specimens
US20080261266A1 (en) * 2004-12-17 2008-10-23 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
US8288121B2 (en) 2004-12-17 2012-10-16 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
US8512978B2 (en) 2004-12-17 2013-08-20 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
US8652803B2 (en) 2004-12-17 2014-02-18 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
WO2007084429A2 (en) * 2006-01-13 2007-07-26 Ventana Medical Systems, Inc. Biological sample processing composition and method
US20070172911A1 (en) * 2006-01-13 2007-07-26 Michael Farrell Biological sample processing composition and method
WO2007084429A3 (en) * 2006-01-13 2007-11-22 Ventana Med Syst Inc Biological sample processing composition and method
AU2007207685B2 (en) * 2006-01-13 2011-12-15 Ventana Medical Systems, Inc. Biological sample processing composition and method
CN111175102A (zh) * 2020-01-13 2020-05-19 沈阳农业大学 一种芍药属植物根尖染色体制片的方法

Also Published As

Publication number Publication date
JPS552988A (en) 1980-01-10
GB2023871B (en) 1982-08-11
FR2428853B1 (de) 1985-04-19
CH641726A5 (de) 1984-03-15
IT1117185B (it) 1986-02-17
BR7903793A (pt) 1980-02-05
NL7904721A (nl) 1979-12-18
DE2826363A1 (de) 1979-12-20
BE876970A (nl) 1979-12-14
DE2826363C2 (de) 1984-08-16
IT7949421A0 (it) 1979-06-14
GB2023871A (en) 1980-01-03
SE7905297L (sv) 1979-12-17
FR2428853A1 (fr) 1980-01-11

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